Protein hydrolysates are being used in numerous food products. Traditionally protein hydrolysates were produced by acid hydrolysis, but today enzymatic hydrolysis is regarded as an attractive alternative.
One of the main problems of protein hydrolysates is that they often taste bitter. When using e.g. soy protein or casein, which are rich in hydrophobic L-amino acids, as the protein source, the protein hydrolysate tends to have bitter taste. In general it is believed that whether the taste of proteins is bitter or not depends on the average hydrophobicity of the L-amino acid residues, such as valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan.
A vast number of enzymes exhibiting peptidase activity are capable of performing enzymatic hydrolysis on vegetable, yeast and/or animal proteins, resulting in highly nutritious protein hydrolysates useful as food additives in products such as soups, sauces, gravies, paste, tofu, bouillon, seasonings, baby formulas, snacks, ready-to-eat meals etc.
Peptidases
All peptidases or proteases are hydrolases which act on proteins or its partial hydrolysate to decompose the peptide bond.
EP 427,385 (The Japanese R & D Association) discloses a genomic gene encoding an alkaline protease derived from yellow molds such as Aspergillus oryzae.
JP-0-2002374 and JP-0-2002375 (Shokuhin), describes an alkaline protease derived from Aspergillus oryzae for use in medicine, food, and detergents.
SU-891777 (Khark) concerns a microbial protease from Aspergillus oryzae, which can be used in food, medicine etc.
JP-5-4035283 (Ajinomoto KK) discloses preparation of enzymes from e.g. Aspergillus oryzae exhibiting endopeptidase activity, which can hydrolyse proteins almost completely.
WO 94/25580 (Novo Nordisk A/S) describes a method for hydrolysing vegetable or animal protein by incubating with a proteolytic enzyme preparation derived from a strain of Aspergillus oryzae.
Aminopeptidases
A subgroup of peptidases (proteases) are called aminopeptidases and are classified under the Enzyme Classification number E.C. 3.4.11 (aminopeptidases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)).
Aminopeptidases are capable of removing one or more amino terminal residues from polypeptides.
JP-7-5034631 (Noda) discloses a leucine aminopeptidase derived from yellow koji mold, which includes Aspergillus oryzae.
JP-7-4021798 (Zaidan Hojin Noda Sangyo) describes the production of miso by adding of a leucine aminopeptidase II prepared by cultivating a number of molds, including Aspergillus oryzae strain 460 and strain IAM 2616.
Van Heeke et al., Bioch. Biophys. Acta, (1992), 1131, 337-340, have disclosed the cloning of a 30 kDa aminopeptidase from the bacteria Vibrio proteolyticus deposited at the American Type Culture Collection under the ATCC No. 15338.
Aspergillus oryzae 460 is known to produce a number of leucine aminopeptidases. The molecular weight of three of these was calculated to 26,500, 56,000 and 61,000, respectively determined by gel filtration (Nakada et al., Agr. Biol. Chem, (1972), 37(4), 757-765; Nakada et al., Agr. Biol. Chem, (1972), 37(4), 767-774; Nakada et al., Agr. Biol. Chem, (1972), 37(4), 775-782). The Aspergillus oryzae 460 strain is deposited at the American Type Culture Collection as A. oryzae (AT7C no. 20386).
Reduction of bitter taste of protein hydrolysates
EP 65,663 and EP 325,986 (Miles Inc.) concerns enzymatic hydrolysis of proteins using a mixture of enzymes containing Aspergillus oryzae derived proteases. The obtained protein hydrolysate has a bland, non-bitter taste.
JP47029577 (Asahi Electro-chemical Co.) concerns a protease, derived from Aspergillus oryzae, which does not produce any bitter component when decomposing protein.
Prior art discloses a plethora of enzymes exhibiting peptidase, aminopeptidase and other enzyme activities. Said enzymes may be derived from a number of microorganisms, including the fungus species Aspergillus oryzae.
In general products, useful for producing protein hydrolysates without a bitter taste, comprise a mixture of peptidase and aminopeptidase activities.
It would therefore be desirable to be able to provide a single-component enzyme (i.e. substantially without any side activity) exhibiting only an activity useful for reducing the bitterness of protein hydrolysates used in food products.